The Staphylococcus aureus CamS lipoprotein is a repressor of toxin production that shapes host-pathogen interaction

Lipoproteins of the opportunistic pathogen Staphylococcus aureus play a crucial role in various cellular processes and host interactions. Consisting of a protein and a lipid moiety, they support nutrient acquisition and anchor the protein to the bacterial membrane. Recently, we identified several processed and secreted small linear peptides that derive from the secretion signal sequence of S. aureus lipoproteins. Here, we show, for the first time, that the protein moiety of the S. aureus lipoprotein CamS has a biological role that is distinct from its associated linear peptide staph-cAM373. The small peptide was shown to be involved in interspecies horizontal gene transfer, the primary mechanism for the dissemination of antibiotic resistance among bacteria. We provide evidence that the CamS protein moiety is a potent repressor of cytotoxins, such as α-toxin and leukocidins. The CamS-mediated suppression of toxin transcription was reflected by altered disease severity in in vivo infection models involving skin and soft tissue, as well as bloodstream infections. Collectively, we have uncovered the role of the protein moiety of the staphylococcal lipoprotein CamS as a previously uncharacterized repressor of S. aureus toxin production, which consequently regulates virulence and disease outcomes. Notably, the camS gene is conserved in S. aureus, and we also demonstrated the muted transcriptional response of cytotoxins in 2 different S. aureus lineages. Our findings provide the first evidence of distinct biological functions of the protein moiety and its associated linear peptide for a specific lipoprotein. Therefore, lipoproteins in S. aureus consist of 3 functional components: a lipid moiety, a protein moiety, and a small linear peptide, with putative different biological roles that might not only determine the outcome of host–pathogen interactions but also drive the acquisition of antibiotic resistance determinants.


Introduction
The Gram-positive bacterium Staphylococcus aureus is a frequent colonizer of the skin and mucosae of the human host [1,2] and is often the cause of skin and soft tissue infections (SSTIs).However, after gaining access to deeper tissues, S. aureus can also cause a variety of more severe diseases, including sepsis, endocarditis, pneumoniae, and osteomyelitis [3,4].In order to survive this versatile lifestyle and changing environmental conditions, S. aureus needs to sense and react to different environmental cues and regulate a defined set of virulence factors, among which surface proteins play a preeminent role [5].It is therefore not surprising that the bacterial surface proteome is dominated by proteins that facilitate interaction with the environment, many of which are lipoproteins (Lpp) [6].Lpp, present in all bacteria, are anchored to bacterial membranes through their N-terminal lipid moiety [7,8].Most research studies characterize 2 components of bacterial Lpp, the protein and the lipid moiety.Due to their localization at the interface of the extracellular environment and the bacterial surface, the protein moiety of S. aureus Lpp can have diverse binding and enzymatic activities [9,10], while the lipid part is a ligand for Toll-like receptor 2 (TLR2) and crucial for immune modulation [11][12][13][14].A third component, a small linear peptide derived from the secretion signal sequences of Lpp precursors, has only been studied in a few bacterial species [15][16][17].
Although several S. aureus Lpp are associated with transporters and contribute to virulence [18], the vast majority of staphylococcal Lpp have not been investigated.In this study, we characterize the CamS Lpp and show that it is a repressor of toxin production in S. aureus USA300, the current epidemic methicillin-resistant S. aureus (MRSA) lineage in the United States [19].This represents a novel biological role that is distinct from the involvement of its linear peptide staph-cAM373 in horizontal gene transfer (HGT) [20].Using mutational analysis, we demonstrate that CamS is a potent repressor of genes encoding cytotoxins, such as α-toxin and leukocidins (lukA, lukB, hlgA, hlgB, hlgC).Mutation of the protein component of CamS increases S. aureus hemolysis of rabbit red blood cells and cytotoxicity towards human polymorphonuclear leukocytes.We show that CamS-mediated gene regulation is critical for S. aureus pathogenesis and mutation of camS leads to a significant increase in virulence in a murine SSTI as well as an in vivo sepsis model.Our discoveries provide evidence for distinct biological functions of an Lpp protein moiety and its associated linear peptide.In addition, the here presented study serves as a starting point for future investigations into the interplay between the newly characterized virulence-associated Lpp CamS with other regulatory systems in S. aureus.

CamS represses S. aureus virulence factors
To delineate the biological role of CamS, we used an in-frame camS deletion mutant (ΔcamS) in the MRSA USA300 wild type (WT) background, a truncated CamS mutant lacking 323 amino acids of the CamS protein moiety (camS Δ69-391 ), and a chromosomal complementation of the camS mutant (ΔcamS::camS) (Fig 1A).The camS Δ69-391 mutant and the WT produced similar amounts of staph-cAM373 (S1A Fig) , as assessed by a previously described Enterococcus faecalis aggregation assay [16].To determine the transcriptional impact of CamS, we performed RNA-sequencing (RNA-seq) of MRSA USA300 WT, ΔcamS, camS Δ69-391 , and ΔcamS::camS cultures grown to late-stationary phase in which the predicted camS promoter (S1Bi Fig) showed high activity (S1Bii Fig) .Genes that showed 2-fold higher or lower expression compared to WT and had an adjusted P value below 0.01 were considered differentially expressed in a CamS-dependent manner.Differential gene expression analysis comparing ΔcamS or camS Δ69-391 with the WT showed an altered transcriptional response of several PLOS BIOLOGY  ).Similar results were obtained when comparing the ΔcamS mutant with ΔcamS::camS (S1 Table ).Among the genes up-regulated in the camS mutants, we detected genes encoding toxins that are associated with staphylococcal virulence and human infection, such as α-toxin (hla) and several leukocidins (lukA, lukB, hlgA, hlgB, hlgC) (Fig 1B).While several other genes were also differentially regulated, including genes associated with metabolism and transport, we decided to focus on characterizing the CamS-mediated repression of S. aureus toxins.
We then validated the regulation of distinct toxins with transcriptional reporters.Construction of promoter fusion plasmids was based on predicted transcription start sites [21] or transcriptomic data from our laboratory.The activity of the hla promoter, fused to a green fluorescent protein reporter gene (P hla -sGFP), was substantially higher in ΔcamS, compared to the WT and ΔcamS::camS, confirming that CamS represses hla (Fig 1Ci).To further understand whether the protein moiety or linear peptide moiety of the CamS Lpp was responsible for the observed effect, site-directed mutagenesis was used to exchange 1 or 2 amino acid residues within the staph-cAM373 sequence ("AIFILAA") to produce inactive linear peptides that did not induce staph-cAM373-mediated E. faecalis aggregation (camS F13A,I14V , camS L15V ) (S1A Fig) .None of the strains showed differences in bacterial growth under the conditions used (S1Ci and S1Cii Fig) .In this extended strain set, expression of the P hla -sGFP fusion was approximately 2-fold stronger after 24 h in the ΔcamS and camS Δ69-391 mutants, compared to the WT and ΔcamS::camS (Fig 1Cii).In addition, both peptide mutants showed hla promoter activities similar to the WT (Fig 1Cii), suggesting that the protein moiety of CamS represses hla.The S. aureus exoprotein (Sae) two-component system (TCS), consisting of the SaeR response regulator, its cognate sensor kinase SaeS, and the 2 accessory proteins SaeP and SaeQ [22,23], was shown to activate hla transcription by binding to the consensus SaeR-binding site upstream of the hla promoter [24,25].As expected, measuring the hla promoter activity in the saePQRS whole gene locus mutant (ΔsaePQRS) confirmed that expression of hla is SaeRS dependent.Deletion of camS in the ΔsaePQRS background (ΔsaePQRS ΔcamS) showed no significant difference in hla promoter activity compared to ΔsaePQRS (Fig 1Cii) demonstrating that hla repression by CamS is dependent on its prior activation by SaeRS.
As shown in Fig 1Di, the lukAB promoter activity (lukAB promoter sequence fused to a red fluorescent protein reporter gene; P lukAB -DsRed) showed a stronger increase over 12 h in ΔcamS compared to the WT and ΔcamS::camS.We also measured the expression of the P lukAB -DsRed fusion after 24 h in an extended strain set, demonstrating that expression of lukAB was significantly stronger in the ΔcamS and camS Δ69-391 backgrounds, compared to the WT and ΔcamS::camS (Fig 1Dii).Similar to our hla promoter study, the lukAB promoter activities in the 2 peptide mutant strains were comparable to the WT (Fig 1Dii).Since the transcription of leukocidins is also regulated by SaeRS [24], we measured the lukAB promoter activity in a ΔsaePQRS mutant as well as the ΔsaePQRS ΔcamS double mutant.Both mutant strains showed no P lukAB -DsRed promoter activity after 24 h (Fig 1Dii).The γ-hemolysin locus consists of 3 genes, hlgA with its own promoter, and the hlgC and hlgB operon [26].We measured activity of both promoter fusions, P hlgA -DsRed and P hlgCB -DsRed in WT, ΔcamS, and camS Δ69-391 as well as in ΔsaePQRS and the ΔsaePQRS ΔcamS double mutant.Similar to promoter activities of hla and lukAB, the activities of the hlgA and hlgCB promoters were significantly higher in strains lacking the CamS protein moiety (ΔcamS and camS Δ69-391 ) and very low or undetectable in the ΔsaePQRS single mutant and the ΔsaePQRS ΔcamS double mutant (Fig 1Ei and 1Eii).
The camS gene is conserved in the genomes of several S. aureus strains (S1D Fig) , and similar camS expression was observed with a camS promoter fusion (P camS -DsRed) in the USA400 MW2 and USA300 LAC strains (S1Ei Fig) .Previous in vitro transcription studies showed that USA300 isolates expressed hla at much higher levels than USA400 isolates, including strain MW2 [27].Our promoter fusion results confirm the much lower in vitro hla expression in USA400 compared to USA300 (S1Eii and S1Eiii Fig) .Similar to USA300, deletion of camS in the USA400 genetic background leads to increased hla promoter activity (P hla -DsRed) compared to the WT (S1Eii and S1Eiii Fig) .Collectively, our in vitro data showed that the disruption of the protein moiety of CamS leads to the derepression of several toxins and suggest that CamS contributes to S. aureus virulence regulation across lineages.

CamS-mediated toxin regulation contributes to S. aureus virulence in vivo
Consistent with up-regulation of hla in strains lacking the camS protein moiety (Fig 1Cii), we detected elevated hemolysis of rabbit red blood cells (RBC) in the presence of stationary-phase culture filtrates from ΔcamS and camS Δ69-391 relative to the WT strain (Fig 2A and S2 Table ).Comparable to our hla promoter activity studies, ΔcamS::camS and both peptide mutant strains (camS F13A,I14V and camS L15V ) showed hemolytic activity similar to the MRSA USA300 WT.Culture filtrates of the hla mutant (hla::FNƩ) revealed that α-toxin is essential for the observed RBC lysis under the tested conditions and simultaneous inactivation of hla and camS (hla::FNƩ ΔcamS) phenocopied the hla single mutant (Fig 2A).
Based on our in vitro data, we hypothesized that mutation of the CamS protein moiety would translate to increased S. aureus virulence in vivo.To determine the role of CamS in murine skin infection, animals were subcutaneously injected with MRSA USA300 WT and mutant strains.Similar to the hla mutant strain (hla::FNƩ), the hla::FNƩ ΔcamS double mutant lacked the capacity to form skin lesions in our murine SSTI model (S2Ai-S2Aiii Fig) , confirming the predominant role of α-toxin to establish cutaneous infections [28,29].At day 6 postinfection, no significant difference in weight loss was observed in mice infected with any of the 4 strains (S2Aiv Fig) .However, mice infected with ΔcamS and camS Δ69-391 showed significantly higher lesion sizes compared to the WT or ΔcamS::camS, suggesting that the protein moiety of CamS also represses hla expression in vivo (Fig 2Bi).To investigate whether the more severe skin lesions were associated with increased bacterial burden in the local skin tissue, skin biopsies were collected on day 6 postinfection and homogenized to enumerate colony-forming units (CFU counts per gram tissue).No significant difference in the bacterial load was observed comparing the 4 strains (Fig 2Bii).We hypothesized that the observed increase in lesion size produced by ΔcamS and camS Δ69-391 is likely due to higher α-toxin production.Subsequently, we examined the amount of α-toxin (Hla) in tissue homogenates at day 6 by dot immunoblot (Figs 2C and S2B).A substantial increase of Hla was detected in tissue homogenates of mice infected with ΔcamS and camS Δ69-391 compared to WT and ΔcamS::camS.Based on our results, we conclude that the CamS protein component acts as a repressor of α-toxin production in our murine SSTI model.
Humans are more sensitive to a wider range of S. aureus toxins than mice [30,31].To gain a deeper understanding of the extent of CamS-mediated virulence regulation, we also investigated S. aureus-mediated killing of human polymorphonuclear leukocytes (hPMN), representing the first line of defense against S. aureus infections [32].To evaluate the contribution of each up-regulated leukocidin (  further demonstrate that the observed hypercytotoxic phenotype was due to the inactivation of CamS, a camS deletion was introduced into the leukocidin transposon mutants (lukA, hlgA, hlgB, hlgC).Mutation of camS in the lukA and hlgC mutant backgrounds resulted in a significant increase in cytotoxicity compared to the lukA and hlgC single mutant strain, respectively.No significant increase in cytotoxicity was observed upon introduction of the camS mutation in the hlgA and hlgB mutant backgrounds, demonstrating that CamS-driven repression mainly affects HlgAB-mediated cytotoxicity in our hPMN killing experiment.
Although S. aureus predominantly results in SSTI, local infections allow access to deeper tissues from where S. aureus enters the bloodstream, leading to bacteremia and the development of sepsis [33].Recently, it was shown that leukocyte-bacteria interactions determine sepsis outcome [34], and we hypothesized that CamS-mediated toxin repression might impact progression and outcome of sepsis.The virulence of the MRSA USA300 WT and the ΔcamS mutant were compared in a 3-day murine sepsis model of infection.After mice were inoculated with equal amounts of the 2 strains, virulence was assessed by monitoring murine survival and weight loss of the infected animals.Mice infected with ΔcamS showed a trend of decreased survival over the course of the experiment (Fig 2Ei).At day 2 postinfection, mice infected with ΔcamS displayed a significant weight loss compared to the mice infected with the WT (Fig 2Eii).No differential colonization of organs (liver, kidney) could be detected between the 2 strains (S2Ci and S2Cii Fig) .The increased mortality rate and weight loss in animals infected with ΔcamS suggests that the up-regulation of toxins in this mutant leads to increased virulence in our murine sepsis model.

Discussion
Despite a wealth of knowledge on virulence factor regulation in S. aureus, we are just beginning to explore the regulatory networks involving Lpp components in S. aureus.Recently, we identified the processing and secretion machinery for Lpp-derived linear peptides in S. aureus [16].The small linear peptide staph-cAM373, derived from the secretion signal sequence of the Lpp encoded by camS, shares homology with a linear peptide in E. faecalis that regulates conjugal plasmid transfer [20].While staph-cAM373 was shown to induce interspecies HGT between E. faecalis and S. aureus in vitro [20], the 43-kDa CamS protein moiety has not been studied yet.By combining transcriptomics, bacterial genetics, functional in vitro assays and in vivo studies, we demonstrate that the CamS protein moiety acts as a repressor of α-toxin (hla) and several leukocidins (lukA, lukB, hlgA, hlgB, hlgC).The loci encoding the CamS-regulated toxins are part of the S. aureus core genome and are present in approximately 99% of sequenced S. aureus strains [35][36][37][38], suggesting that CamS-mediated virulence suppression is likely conserved in the species S. aureus.Indeed, we showed that the deletion of camS in another S. aureus lineage, USA400, also results in the transcriptional up-regulation of α-toxin.The abovementioned toxins are all under the regulatory control of the Sae TCS in S. aureus [24,25] and mutation of sae leads to the loss of observable toxin repression by CamS.Some bacterial Lpp were shown to be an integral part of TCSs and/or interact with kinases and thereby modulate their regulatory activity [23,39,40].A mechanism that might also underlie CamS-mediated transcriptional repression of toxins.
Although α-toxin is capable of lysing various cell types, rabbit erythrocytes exhibit greater sensitivity to α-toxin exposure compared to human erythrocytes, attributed to the absence of the high-affinity toxin receptor ADAM10 (A Disintegrin and Metalloproteinase 10) on human RBC [41].Accordingly, we used rabbit RBC in our experimental setup to capture the full extent of CamS-mediated α-toxin repression.Our findings indicate that the up-regulation of α-toxin, mediated by the deletion of the CamS protein moiety, results in enhanced lysis of rabbit RBC.We also addressed herein the role of CamS-mediated killing of hPMN.MRSA USA300 can produce up to 5 different pore-forming, bicomponent leukocidins (γ-hemolysins HlgAB and HlgCB, LukSF-PV/PVL, LukAB/HG, and LukED) with the ability to lyse hPMN [38,42].We did not observe differential expression of the leukocidins lukSF-PV and lukED in our RNA-seq analysis, but culture broth-dependent expression of both leukocidins were reported in MRSA USA300 [31].A lukA mutant strain only showed a slight reduction in cytotoxicity towards hPMN compared to the WT strain, despite lukA and lukB being the 2 most highly CamS-repressed leukocidins in our transcriptomics data.This discrepancy is most likely explained by previous findings, demonstrating that LukAB is primarily associated with the bacterial cell during stationary phase when grown in rich media [43,44].Therefore, despite the elevated expression and promoter activity of lukAB in the camS mutant strain, other secreted leukocidins might have a stronger impact on hPMN cytotoxicity under our experimental conditions.Indeed, deletion of the γ-hemolysins hlgA, hlgB, or hlgC resulted in minimal cytotoxic activity towards hPMN.Based on our results and the bicomponent nature of HlgAB and HlgCB, the increased cytotoxicity of the camS mutant strain seems to be mainly driven by derepression of the HlgAB toxin under our experimental conditions.
Our in vitro data on CamS-mediated toxin repression were reflected in two different in vivo models of infection.MRSA USA300 is the predominant cause of SSTIs [45] with α-toxin contributing to superficial and invasive disease and disturbing host immunity during skin infections and recurring disease [46,47].By using camS mutant strains, either lacking the CamS protein moiety, a functional staph-cAM373 peptide or both, we demonstrate that the absence of the CamS protein moiety, but not staph-cAM373, leads to significantly increased lesion size in our infection model.We also tested the importance of CamS in a sepsis model of infection, and we observed a clear trend in increased mortality and weight loss in animals infected with a camS mutant strain.It was previously shown, that γ-hemolysins contribute to virulence during systemic infection [48] and septic arthritis [49].However, murine neutrophils lack the responsive receptors for HlgAB and HlgCB [31], and the impact of CamS-mediated leukocidin repression might not be fully captured in our murine sepsis model.Future research involving a systemic infection model in rabbits, which are more sensitive to infection by S. aureus, is needed to provide detailed insights into CamS-mediated virulence regulation in bacteremia and sepsis.
Our results show that the linear peptide and the protein moiety of a S. aureus Lpp can have distinct biological functions.With widespread camS representation in staphylococcal genomes, further studies will show whether CamS has additional roles in different S. aureus strains and lineages.Our RNA-seq analysis revealed that CamS regulates not only genes encoding toxins but also other important virulence factors, including the serine protease-like (Spl) proteases.It remains to be seen whether CamS interacts with components of known global virulence regulatory systems to fine-tune S. aureus virulence and host-pathogen interaction.

Ethics statement
All animal work was approved by and performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz Medical Campus under protocol number #00486 and #1137.

Bacterial strains and plasmids
Bacterial strains and plasmids are listed in S3 Table, and primers are listed in S4 Table .DNA sequencing of the constructed plasmids was performed at the Molecular Biology Service Center at the University of Colorado Anschutz Medical Campus.If not specified otherwise, S. aureus and E. coli were cultured in tryptic soy broth (TSB) at 37˚C with shaking at 250 rpm.Where appropriate, antibiotics were added to the media at the following final concentrations: chloramphenicol (Cm), 10 μg mL −1 ; erythromycin (Erm), 5 μg mL −1 .E. coli strains with plasmids were maintained in media supplemented with ampicillin (Amp) at 100 μg mL −1 .

Mice
Seven-week-old male and female BALB/cJ mice (Jackson Laboratories, RRID: IMSR_JAX:000651) and 7-week-old female C57BL/6J mice (Jackson Laboratories, RRID: IMSR_JAX:000664) were purchased from the Jackson Laboratories and were housed in specific pathogen-free facilities at the University of Colorado Anschutz Medial Center Animal Facility.Mice were allowed to acclimate for 1 week prior to experimentation.At experimental endpoints, mice were killed via CO 2 inhalation followed by cervical dislocation.

Human polymorphonuclear leukocytes (hPMN)
Human adult polymorphonuclear leukocytes from healthy blood donors were purchased from the Division of Pulmonary, Critical Care and Sleep Medicine at the National Jewish Health at Denver, where they were isolated by the plasma-Percoll method [50].
Genomic DNA of S. aureus strains was isolated using the Puregene cell kit (QIAGEN; Cat#158567), including a lysis step with lysostaphin (100 ng/μL, ABMI Products LLC, Cat#LSPN).A marker less deletion of camS in MRSA USA400 MW2 (MW1844) was constructed as previously described for deleting camS (SAUSA300_1884) in the MRSA USA300 background (LAC* ΔcamS) [16].Briefly, regions flanking the gene were amplified from MRSA USA400 MW2 genomic DNA (AH843) using primers KAS26/KAS27 and KAS28/KAS29.The amplified products were column purified, digested with EcoRI/XhoI and XhoI/SalI (New England Biolabs) and ligated into pJB38, digested with EcoRI and SalI, to generate pKAS57.The plasmid was electroporated into E. coli DC10B, sequenced (primers KAS169, KAS170, KAS41, KAS42) and subsequently electroporated into MRSA USA400 MW2, as previously described [53,54].Deletions were generated as described in [55], and mutants were confirmed by PCR with primers KAS33/KAS34.The previously constructed plasmid pKAS07 (pJB38 with approximately 1,000 bp regions flanking the camS gene) [16] was used to introduce a camS deletion into LAC* ΔsaePQRS.The deletion was generated as previously described [55] and confirmed by PCR with primers KAS33/KAS34.Double mutants in camS and leukocidins were generated by introducing leukocidin transposon mutations into the ΔcamS background using bacteriophage transduction as described above.
The camS mutation in the LAC* background (LAC* ΔcamS) was complemented by introducing the camS gene at its original location on the chromosome.The camS complementation strain (LAC* ΔcamS::camS) was constructed by amplifying the camS gene and flanking regions from the MRSA USA300 LAC* genome (AH1263).First, the camS gene and a region covering approximately 600 bp upstream of camS were amplified with primers KAS266 and KAS281, column purified, digested with SalI/EcoRI and ligated into pJB38 digested with the same restriction enzymes.The resulting plasmid was electroporated into E. coli DC10B.After propagation, the plasmid was digested with XhoI/EcoRI and ligated with a DNA fragment located downstream of the camS gene, which was amplified with primers KAS268 and KAS269 and digested with XhoI/EcoRI to generate pKAS61.The resulting complementation construct carries an XhoI restriction site located 43 nt downstream of the camS stop codon that serves as a watermark for the complemented strain.Introduction of pKAS61 into E. coli DC10B, transfer to LAC* ΔcamS, selection for complemented strains, and plasmid sequencing was carried out as described for construction of the camS mutant in MRSA USA400 MW2.
A camS mutant strain with a truncated camS gene was constructed (LAC* camS Δ69-391 ).The expressed truncated CamS protein carries an intact signal peptide sequence that encodes for the staph-cAM373 linear peptide but lacks 323 amino acids (aa) of the protein moiety of CamS.Briefly, DNA fragments (approximately 1,000 bp in size) flanking the region targeted for deletion (nt 205 to nt 1,173 of the camS gene) were amplified from MRSA USA300 LAC* genomic DNA (AH1263) using primers KAS270/KAS282 and KAS283/KAS272.The product amplified with primers KAS270/KAS282 was digested with EcoR/SalI and ligated into pJB38 digested with the same enzymes.The resulting plasmid was transferred into E. coli DC10B by electroporation.After propagation, the plasmid was digested with SalI/NheI and ligated with the DNA fragment amplified with primers KAS283/KAS272 and digested with the same enzymes to generate pKAS63.Introduction of the resulting plasmids into E. coli DC10B, transfer to LAC* WT, selection for mutated clones, and plasmid sequencing was carried out as described for construction of the camS mutant in MRSA USA400 MW2.
The allelic exchange plasmid pJB38 carrying camS was utilized as template to exchange amino acids within the staph-cAM373 peptide sequence.In brief, a DNA fragment covering the camS gene was PCR amplified from MRSA USA300 LAC* genomic DNA (AH1263) using primers KAS192 and KAS193, digested with restriction enzymes EcoRI and SalI and ligated into pJB38, resulting in plasmid pKAS38.This plasmid was transferred into E. coli Top10 by electroporation and sequenced with primers KAS169/KAS170.Site-directed mutagenesis was performed using the QuickChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Cat#200523) with the modified method as recommended by Liu and Naismith [56] with primers KAS194/KAS195 and KAS274/KAS275, resulting in plasmids pKAS39 and pKAS53, respectively.Plasmid pKAS39 has a double nucleotide exchange in a phenylalanine and isoleucine codon to an alanine and valine codon, respectively (F13A/I14V).The plasmid pKAS53 has a single valine substitution in place of a leucine (L15V) in the staph-cAM373 peptide amino acid sequence.The mutated plasmids were transferred into E. coli DC10B by electroporation and sequenced with the primer KAS44.Both plasmids were then electroporated into MRSA USA300 LAC* WT (AH1263) and mutations in the chromosome were generated as described in [55].Mutations in the S. aureus genome in both strains LAC* camS F13A,I14V and LAC* camS L15V were verified by sequencing the amplified PCR product (KAS26/KAS29) using primers KAS175, KAS83, KAS28, KAS44, KAS65, KAS192, and KAS193 from the mutants genome.Numbering corresponds to CamS amino acid sequence.

Construction of promoter fusion plasmids
Construction of promoter fusion plasmids was based on predicted promoter sequences from Prados and colleagues [21] or promoter predictions using RNA-seq data from [57] and this study.The hla promoter-sGFP transcriptional reporter (P hla -sGFP) was generated by amplification of the putative promoter region of hla (SAUSA300_1058) from MRSA USA300 LAC* genomic DNA (AH1263) using primers CLM429/CLM430, digested with HindIII and KpnI before ligation in the shuttle vector pCM11.The hla promoter region fragment fused to sGFP was amplified from the vector with primers CLM463/CLM332, digested with NheI and EcoRI, and ligated into vector pCM28, resulting in pCM36.An hla promoter-DsRed transcriptional reporter (P hla -DsRed) in MRSA USA300 LAC* and MRSA USA400 MW2 was generated in the shuttle vector pHC48, which expresses DsRed under the control of the S. aureus sarA P1 promoter [58].A fragment containing the putative promoter of hla was amplified from MRSA USA300 LAC* (AH1263) and USA400 MW2 (AH843) genomic DNA using primers KAS450/ KAS451, digested with XbaI and KpnI, and subsequently ligated upstream of the DsRed gene in pHC48, where the sarA promoter fragment was excised using the same enzymes resulting in the reporter plasmid pKAS108 for MRSA USA300 and pKAS109 for MRSA USA400.A camS promoter-DsRed transcriptional reporter (P camS -DsRed) was generated in the shuttle vector pHC48 as described for the hla promoter fusion.A fragment containing the camS promoter was amplified from MRSA USA300 LAC* genomic DNA (AH1263) using primers KAS175 and KAS193, digested with SalI and KpnI, and subsequently ligated upstream of the DsRed gene in pHC48, where the sarA promoter fragment was excised using the same enzymes resulting in the reporter plasmid pKAS44.Transcriptional reporters for lukA (P lukA -DsRed), hlgA (P hlgA -DsRed), and hlgCB (P hlgCB -DsRed) were constructed in a similar fashion with primers KAS418/KAS419 (P lukA ), KAS446/KAS447 (P hlgA ), and KAS448/KAS449 (P hlgCB ) resulting in the reporter plasmids pKAS92, pKAS103, and pKAS105, respectively.A control strain harboring pHC48 with a promoterless DsRed gene was constructed by excising the sarA promoter with BamHI and religation of the plasmid (pKAS43).All transcriptional fusion plasmids were transferred into E. coli DC10B by electroporation [54], sequenced with primers KAS113 and KAS116, and subsequently electroporated into MRSA USA300 LAC* WT, MRSA USA400 MW2 WT, and their respective mutant derivatives.

Bacterial growth kinetics and yields
To assess growth kinetics of S. aureus WT and mutant strains, 200 μL of diluted cultures with a starting optical density at 600 nm (OD 600 ) of 0.02 in TSB were grown in a clear 96-well microplate and absorbance was measured every 30 min with a Synergy H1 Microplate Reader (Bio-Tek) for 24 h (37˚C, continuous orbital shaking).Bacterial growth yields were assessed by counting CFU on tryptic soy agar (TSA) from cultures grown for 24 h at 37˚C with agitation at 250 rpm.

Aggregation assay
To assess the production of the linear peptide staph-cAM373 in S. aureus culture filtrates, we employed a previously reported cell aggregation assay [16,59].Briefly, E. faecalis JH2-2 cells containing a peptide-responsive pAM373::Tn918 plasmid were grown overnight in TSB at 37˚C with 10 μg/mL tetracycline at 250 rpm shaking, washed twice, and resuspended to an OD 600 of 0.2 in chemically defined medium (CDM) [60].Culture filtrates from S. aureus WT and mutant strains, grown in CDM until reaching an OD 600 of 3, were mixed in a 1:1 ratio with E. faecalis cells in a round-bottom glass tube and incubated at 37˚C in a shaking incubator (250 rpm) for 4 ½ h.Cell aggregation was quantified by measuring the turbidity of the suspension with a MicroScan turbidity meter compared to that of the medium.

Reporter assays
To assess promoter activity, overnight cultures grown with the appropriate antibiotic (37˚C, 250 rpm shaking) were washed twice in phosphate-buffered saline (PBS), diluted to an OD 600 of 0.02 in TSB, and 200 μl were added in triplicate per strain to a 96-well black microplate with clear bottom (Corning Costar, Cat#3603).Plates were incubated at 37˚C with shaking (1,000 rpm) in a humidified microtiter plate shaker (Stuart SI505, Cole-Parmer) and absorbance (A 600 ) and fluorescence intensity for sGFP (Excitation 480 nm, Emission 515 nm) or DsRed (Excitation 549 nm, Emission 588 nm) were measured with a Tecan Infinite M Plex plate reader.For each experiment, fluorescence values from triplicate wells were averaged, set relative to bacterial growth (A 600 ), and the background fluorescence from strains carrying the respective promoterless plasmid (pCM28 or pKAS43) was subtracted.Data are represented as relative fluorescence unit (RFU).

Hemolysis assay
A rabbit RBC hemolysis assay was used to determine hemolytic activity in bacterial culture filtrates from WT and mutant strains as previously described [61].Briefly, culture filtrates were generated from cultures grown in TSB for 24 h at 37˚C with shaking at 250 rpm.RBC from defibrinated rabbit blood (Hemostat Laboratories, Cat#10052-762) were prepared by washing 4 to 5 times with 1.2 × PBS and resuspending the pellet in 1.2 × PBS at 3% (v/v).Bacterial culture filtrates were serially diluted in 2-fold steps in 1.2 × PBS in a 96-well microtiter plate, mixed with the 3% RBC solution (70:30, RBC:culture filtrates) and incubated statically at room temperature (RT) for 1 ½ hours.Hemolysis was assessed by the loss of turbidity measured at OD 633nm using a Tecan Infinite M Plex plate reader.To determine the final concentration (%) of the culture filtrate required for 50% of RBC lysis (EC 50 ), the data were analyzed with Graph-Pad Prism 9 (version 9.4.1.)(4-parameter logistic curve with least squares regression fit).Data are presented as hemolytic activity relative to the median of the WT strain.

Cytotoxicity assay
A modified Giemsa staining kit (Differential Quik III Stain Kit, Electron Microscopy Sciences, Cat#26096) was used to assess the purity of hPMN (>98%) before each assay.For hPMN infection assays, bacterial cultures grown overnight in TSB (37˚C, 250 rpm shaking) were subcultured at a starting OD 600 of 0.02 and grown for 24 h as described above.All bacterial cultures were normalized to the same OD 600 and pelleted by centrifugation at 5,000 × g for 10 min.Supernatants containing exoproteins were collected, filtered using Costar Spin-X 0.2-μm centrifuge tube filters (Sigma-Aldrich, Cat#CLS8160) and stored at −20˚C until usage.S. aureus culture filtrates (10%, v/v) were used to intoxicate hPMN, seeded at 1 × 10 5 cells/well in 100 μl RPMI-1640 (Gibco, Cat#11835030) with 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Cat#S11550) and incubated for 1 h at 37˚C and 5% CO 2 .Following incubation, cells were centrifuged at 300 × g for 10 min, and LDH release was assayed as a measure of hPMN viability using the CyQuant LDH Cytotoxicity Assay (Thermo Fisher, Cat#C20300) according to the manufacturer's instructions.Briefly, 50 μl of cell supernatant was removed and mixed with 50 μl of LDH reagent and incubated for 30 min at RT. S. aureus growth medium (TSB) was used as 100% viability control, and lysis buffer from the CyQuant LDH Cytotoxicity Assay served as control for 100% cell lysis.Bacterial LDH production in culture filtrates without addition of hPMN was assessed, and no bacterial LDH was detectable.Viability of hPMN was measured with a Tecan Infinite M Plex plate reader (Absorbance at 490 nm and 690 nm).Percentage of cytotoxicity was calculated by subtracting background values (background signal from instrument, growth medium) and normalizing to 100% lysis.

Sample preparation for RNA purification
Bacterial overnight cultures (37˚C, TSB, 250 rpm) were diluted to an OD 600 of 0.02 in 5 mL TSB, grown for 24 h at 37˚C with shaking (250 rpm) and 2 mL of each culture were centrifuged at 5,000 × g for 5 min.The pellet of each sample was resuspended in 1.5 mL RNAprotect Bacteria Reagent (QIAGEN, Cat#76506) and incubated and centrifuged according to the manufacturer's recommendations.The pellet was shock frozen in liquid nitrogen, and samples were stored at −80˚C until RNA purification.

RNA purification
RNA was extracted using an RNeasy Mini Kit (QIAGEN, Cat#74104) with the following modifications.Cell pellets were incubated in 100 mM TRIS-HCL with 800 ng/μL lysostaphin for 25 min at 37˚C.Next, 700 μl of RLT buffer containing β-mercaptoethanol was added, and the samples were transferred to lysing matrix B tubes (Fisher Scientific, Cat#MP116911050).After 3 bead beating steps (30 s × 3, with 1 min on ice between each), RNA extraction was followed according to the standard Qiagen RNA extraction protocol.Purified RNA was treated with the Turbo DNA-free Kit (Invitrogen, Cat#AM1907) according to manufacturer's instructions.RNA concentrations were determined with a Nanodrop spectrophotometer (Thermo Fisher Scientific), and RNA quality control for all RNA-seq samples was performed at the Genomics Core at the University of Colorado Anschutz Medical Campus using an RNA ScreenTape resulting in an RNA integrity number (RIN) between 8.9 and 9.2 for all samples.

RNA-seq
Depletion of ribosomal RNA and library preparations were performed at the Genomics Core at the University of Colorado Anschutz Medical Campus using the Illumina Ribo-Zero Plus rRNA depletion kit (Illumina, Cat#20037135) and the Zymo-Seq RiboFree total RNA library kit (Zymo Research, Cat#R3003).Sequencing (150 base pairs, paired-end) was performed on an Illumina NovaSEQ 6000.Raw sequencing reads in fastq format were imported into the CLC Genomics Workbench (QIAGEN, version 20.0.4), adapter sequences were trimmed (automatic removal of read-through adapter sequences, removal of low-quality sequence (limit = 0.05)), and trimmed sequences were mapped to the USA300 FPR3757 reference genome (NC_007793) using Qiagen CLC Genomics Workbench default settings (mismatch cost: 2, insertion cost: 3, deletion cost: 3, length fraction: 0.8, similarity fraction: 0.8).Count tables were prefiltered to keep only rows that have a count of at least 10 and were then analyzed for differential gene expression of samples using RStudio (version 2022.07.0,RRID: SCR_000432) and the R package DESeq2 (version 4.2, RRID:SCR_015687) [62] and visualized with R package pheatmap (version 0.2, RRID:SCR_016418).The threshold for differential expressed genes was set at >1 or <−1 Log2 fold change (L2FC) using an adjusted p-value (pADJ) of 0.01.

Murine SSTI model
To prepare bacterial inocula for infection, MRSA USA300 LAC* WT, ΔcamS, camS Δ69-391 , ΔcamS::camS, hla::FNƩ, and hla::FNƩ ΔcamS were grown overnight in TSB (37˚C, shaking at 250 rpm).The following day, all cultures were subcultured to an OD 600 of 0.05 in fresh TSB and allowed to grow to exponential phase (OD 600 0.4 to 0.6) at 37˚C and shaking at 250 rpm.Bacterial cells were washed and pelleted in PBS and resuspended in PBS to reach an inoculum of approximately 1 × 10 8 CFU in 50 μL, which was verified by colony counting after 24 h of incubation at 37˚C.Mouse infections were performed as previously described [63].Briefly, 1 day prior challenge male or female BALB/cJ mice abdomen were shaved, and residual hair was removed with a 30-s application of Nair Hair Remover Lotion (Church & Dwight).Immediately prior to injection, abdomens were sanitized with alcohol wipes, and bacteria were injected intradermally.Lesion size on skin and mouse body weights were measured before infection and every day thereafter for a period of 6 days.Digital images of the skin lesions were taken using a Canon PowerShot ELPH 180 camera and analyzed with FIJI-ImageJ (NIH) software [64].At day 6 postinfection, the skin lesions were collected with a 12-mm diameter biopsy punch (Fisher Scientific, Cat#NC9253254), weighted, and homogenized in PBS with 1-mm zircona/silica beads (BioSpec Products, Cat#NC19847287) in 3 bead beating steps (1 min × 3, with 1 min on ice between each) with a Mini-Beadbeater-16 (BioSpec).Homogenates were serially diluted in PBS and plated on mannitol salt agar (MSA, BD Cat#211407) supplemented with 5.2 μg/mL cefoxitin to determine CFU counts.

Dot blots
To detect Hla protein in mouse skin lesions, tissue homogenates were centrifuged twice at 5,000 × g for 20 min and normalized to 1 × 10 8 CFU/gram tissue followed by 2-fold dilutions in PBS.Then, 20 μl of the mouse homogenates from SSTI experiments or 20 μl of a 2-fold dilution of recombinant Hla protein from S. aureus (0.5 mg/mL in H 2 O, Millipore Sigma, Cat#H9395) were spotted on a 0.45-μm nitrocellulose membrane (BIO-RAD, Cat#1620117) using a Bio-Dot microfiltration apparatus (BIO-RAD, Cat#1706545).The membrane was blocked overnight with Intercept blocking buffer Tris-buffered saline (TBS, LI-COR, Cat#92760001) containing 5% albumin human serum (Calbiochem, Cat#12667) to prevent nonspecific antibody binding.After blocking, the membrane was incubated for 1 ½ hours with a polyclonal rabbit anti-Hla antibody (gifted by Patrick M. Schlievert) diluted 1:5,000 in Intercept buffer + 0.2% Tween 20 + 1% human serum.The membrane was washed 3 times with TSB containing 0.1% Tween 20 (TBST) and incubated with goat anti-rabbit antibodies (IRDye 800CW conjugated, LI-COR Biosciences, Cat#926-32211) diluted 1:15,000 in Intercept buffer TBS with 0.1% Tween 20 and 1% human serum for 1 h at RT.The membrane was washed 4 times with TBST for 5 min each and then imaged on a LiCor Odyssey CLx Imaging System (LI-COR Biosciences) and analyzed with Image Studio Lite (version 5.2, LI-COR).

In vivo murine sepsis model
Bacterial inoculum for infection was prepared as described for the murine SSTI model with a final inoculum of 1.2 × 10 7 CFU in 100 μL for each bacterial strain, which was verified by colony counting after 24 h of incubation at 37˚C.Female, 7-week-old C57BL/6J mice were then infected via retro-orbital injection of either MRSA USA300 WT or ΔcamS, and the infection was allowed to proceed for 72 h.Mouse weights were recorded at 24-h intervals, and mice were killed with CO 2 if their body weight was below 75% or at the completion of the experiment.For bacterial enumeration, organs (kidneys, liver) were harvested, weighted, and homogenized in PBS as described for the murine SSTI model.Homogenates were serially diluted in PBS and plated on MSA supplemented with 5.2 μg/mL cefoxitin to determine CFU counts.

Generation of dendrogram
The dendrogram of CamS (SAUSA300_1884) orthologs was created based on KEGG SSDB (Sequence Similarity DataBase) database entries [65].The top 50 orthologs of SAUSA300_1884 were determined with the following settings: Show (Best), Threshold (100), Top50, and the dendrogram was created with the "Create Dendrogram (single)" function.